Immunoassay reagents have functionality that can be broken down into two broad components: signal generation (also known as amplification) functionality, and ligand binding functionality. The signal generation functionality is required for detection of ligand binding to analyte, and the ligand binding functionality is the specificity of the reagent for the analyte. The ligand binding activity is accomplished by covalently attaching ligands to the particle surface. Antibodies and small haptens of biological significance, such as thyroxin, are examples of common ligands.
Fluorescent and chemiluminescent compositions have been widely used in signal generation components of immunoassays. A typical signal generation component includes a carrier, such as a latex particle, dyed with a fluorescent or chemiluminescent composition. In particular, metal chelates have been widely used as fluorescent and chemiluminescent dyes because of their generally large stokes' shift, sharp emission peak and long emission wavelength. Typically, the metal chelates are a complex formed of a metal, such as europium, samarium, or terbium, and ligands such as thiophenetrifluorobutanedione (TTA), napthyltriflurobutanedione (NTA), and 4,7-diphenyl-1,10-phenathroline (DPP) trioctyl phosphine oxide (TOPO), triphenyl phosphine oxide (TPPO). Some commonly used metal chelates include Eu(TTA)3DPP and Eu(NTA)3DPP.
Dyed carriers that are used to make assay reagents must have both the capability to provide signal generation, and the chemical functionality for covalent attachment of ligands. The process used to attach the ligands can be critical to the quality of the specificity of the ligand binding functionality. The specificity of the ligands can be compromised in several ways by inappropriate choices of attachment chemistry. For example, if passive adsorption of the ligands occurs simultaneously with covalent attachment, the passively adsorbed ligands may come off of the carrier during the assay. The resulting free ligand will interfere with the assay and reduce its sensitivity. Another problem that can occur is the non-specific binding of the reagent to the other carrier, which leads to elevation of the immunoassay signal in the absence of analyte.
Ligands are often attached to polystyrene particles through carboxy groups that are attached directly to the surface of the particles. This approach can have both of the problems described above. The passive adsorption and nonspecific binding problems can be avoided by introducing one or more layers of immobilized hydrogel polymers, such as an aminodextran, between the polystyrene particles and the ligands. It is important for elimination of adsorption and nonspecific binding to have a continuous layer of these hydrogel polymers.
However, the coating of particles that are dyed with conventional dyes such as Eu(TTA)3DPP or Eu(NTA)3DPP typically result in coating densities of aminodextran that are significantly less than optimal for preventing non-specific binding. The coupling density of the aminodextran is typically reduced by 40-50% from its maximum when these dyes are added at the optimum level for the chemiluminescent response of the reagent. Lower levels of dye give compositions that interfere less with the aminodextran coating process, but reducing the dye content also reduces the chemiluminescence necessary for a strong signal.
Accordingly, there remains a need in the art to provide a more optimal balance between the amplitude of the response signal and the specificity of the signal generation components in an specific binding assay.